The goal of the proposed work is to create a novel gene delivery platform to image and kill human liver cancer, specifically hepatocellular carcinoma (HCC). Current standard of care treatments employ chemotherapy, which is often delivered transarterially to enhance localization of treatment to the tumor tissue. However, transarterial chemotherapy has only proven effective for palliative care, and often results in damage to surrounding healthy liver tissue which can lead to liver failure and death. We propose to address this concern by designing a novel drug delivery system that could enhance tumor killing while reducing damage to healthy cells. We will deliver DNA encoding herpes simplex virus thymidine kinase (HSVtk), and use HSVtk to convert the prodrug ganciclovir (GCV) into a cell-killing drug. HSVtk DNA will be delivered using polymeric nanoparticles which we will engineer to selectively deliver DNA to HCC, thus enabling cancer specific cell killing. We will further promote HCC selectivity by designing novel plasmid promoters that restrict HSVtk expression to HCC cells. We will administer these nanoparticles transarterially using a mouse orthotopic model of human HCC to mimic the minimally invasive surgical technique currently employed in HCC therapy. HSVtk also phosphorylates nucleoside analogs (radiotracers), making them visible via PET imaging, which will enable us to image and monitor the tumor in vivo. Based on preliminary work, we hypothesize that we will be able to engineer both nanoparticles and plasmids that restrict cell killing to human HCC while avoiding off-target effects to human hepatocytes. We hypothesize that this delivery system will enable prolonged survival in an orthotopic human HCC mouse model while simultaneously enabling tumor imaging in vivo. In Specific Aim 1 we will demonstrate cancer- specific gene delivery via nanoparticles to nine human HCC lines as compared to three human hepatocyte cell lines in vitro, and further examine this selectivity in a mouse orthotopic human HCC model. Additionally, we will examine the mechanism by which these nanoparticles exhibit cancer selectivity, which may help to elucidate how cancer-targeting nanoparticles can be improved even further in the future. In Specific Aim 2, we will design a transcriptional promoter that enables HCC-specific gene expression, and deliver this plasmid using nanoparticles to demonstrate enhanced cancer specificity both in vitro and in vivo. In Specific Aim 3, we will demonstrate the efficacy and cancer selectivity of the HSVtk/GCV cell killing system in vitro and in vivo, in addition to using HSVtk and a radiotracer to image human HCC in a mouse orthotopic tumor model. The ultimate goal of this research is to create a cancer-specific, theranostic, and minimally invasive treatment for human HCC.